Urinary citrate is one of the most important known endogenous inhibitors of calcium nephrolithiasis. Urinary citrate excretion is primarily determined by its fractional reabsorption in the proximal tubule. Previously, a major impediment to studies of citrate transport has been the lack of a suitable cell culture model. However, we have now clearly identified a method to study citrate reabsorption in a proximal tubule cell line (see preliminary data). In addition, the recent cloning of an apical dicarboxylate transporter from the proximal tubule by Pajor allows new insight into the regulation of citrate transport. This with our new culture model will allow us to examine several critical issues in the molecular regulation of citrate transport. The specific aims are to characterize both acute and chronic regulation of citrate absorption in the proximal tubule. Acute regulation of citrate transport by pH and divalent cations, in particular will be addressed. Although a variety of previous studies have addressed the influence of pH on citrate transport, our proposed studies will be able to directly address simultaneously the role of intracellular pH (to be measured with fluorescent probes) as well as extracellular pH. In regards to divalent cations, our preliminary data demonstrate dramatic effects of extracellular calcium and magnesium on citrate transport. Our findings suggest that changes in luminal calcium and magnesium concentrations within the ranges seen clinically may be important in determining urinary citrate excretion. The proposed studies will also address chronic regulation of citrate reabsorption. Our cell culture method will allow us to directly examine the mechanism of chronic regulation by pH, hypokalemia, and starvation. We hypothesize that changes in citrate reabsorption with each of these result from changes in the abundance and/or cellular distribution of the citrate transporter. In the proposed studies we will be able to measure the direct in vitro effects of these stimuli on citrate transport in polarized cells, and on mRNA and protein levels and cellular distribution of the dicarboxylate transporter.